This project is focused on the elucidation of molecular signal transduction mechanisms involved in activation of NF-kappaB. It complements project 722-13 and parts of project 431-23, which aim to identify and characterize novel in vivo and in vitro functions of NF-kappaB and its regulators. NF-kappaB denotes a family of dimeric transcription factors. These factors are central to the regulation of both adaptive and innate immune responses. They are rapidly activated by numerous stress- and pathogen-derived signals encountered by a variety of cells. Once activated, these factors are responsible for executing genetic programs such as those required for the defense of the host against pathogenic insults. NF-kappaB factors have additional roles, especially during the development of the immune system. In contrast to such important contributions to health, these factors can also be directly or indirectly involved in many diseases, when they are aberrantly activated. The project is intended not only to lead to a better basic understanding of signaling and regulatory processes, but, in addition, to provide a basis for new approaches to treating diseases. The identification of novel components and mechanisms of signaling pathways involved in control of NF-kappaB activity may provide potential new targets for therapeutic interventions in specific diseases. Diseases in which NF-kappaB makes critical contributions include inflammatory and autoimmune diseases as well as many types of cancer. NF-kappaB may drive inflammatory processes that indirectly support tumor development of epithelial cells, e.g., or it may directly support tumorigenesis by ensuring the afflicted cells survival, or both. Consequently, NF-kappaB and the network of signaling proteins that control the activity of these transcription factors become potential targets for therapeutic interventions. Most inflammatory signals as well as many other stress signals stimulate the classical NF-kappaB activation pathway. In this pathway the inhibitor of kappaB kinase (IKK) complex phosphorylates small inhibitory IkappaB proteins, leading to their proteolytic degradation, thus liberating NF-kappaB to translocate to the nucleus and regulate target genes. We have previously identified an adaptor protein termed Conection to IKK and SAPK/JNK (CIKS) that is associated with the IKK complex. It can activate the classical pathway when overexpressed. We are investigating underlying mechanisms by which CIKS regulates the classical NF-kappaB activation pathway. In addition to the classical pathway of activation, NF-kappaB can also be activated via an alternative or non-classical pathway, independent of the IKK complex. This second pathway involves processing of the p100 precursor form of NF kappaB2 to p52, which leads to the generation of p52/RelB heterodimers that are free to translocate to the nucleus. Based on analyses of B cell defects in NF-kappaB2 knockout mice, we previously identified the TNF family member B cell activating factor (BAFF) as the physiologic inducer of this second activation pathway in B cells. In addition, this pathway is activated by the Lymphotoxin beta Receptor (LTbetaR) in stromal cells, where it contributes importantly to stromal cell-dependent immune functions. We are investigating the mechanisms by which these receptors signal processing of p100 to p52. We have determined that the absence of the TNF receptor-associated factor-3 (TRAF3) results in a constitutive activation of the alternative pathway, suggesting an inhibitory role for TRAF3 in this pathway. We have generated TRAF3 mutants and have begun to determine the domains necessary for inhibition of the alternative pathway and the domains required for activating signals to reach and neutralize TRAF3. We are using FRET technology to assay for interactions of signaling proteins that are likely to be involved in the processing pathway. We are also using this technology to probe interactions of the Bcl-3 regulatory protein with chromatin-associated factors. Bcl-3 is known to increase NF-kappaB activity and may do so by serving as a bridge between NF-kappaB factors and transcriptional co-activators within chromatin. We are also investigating the signaling pathways involved in activation of NF-kappaB2 and Bcl-3 during establishment of central (thymic) immunologic tolerance to self. As described in project 722-13 we have shown that these proteins serve a partially redundant function in thymic stroma that is critical for negative selection of autoreactive T cells. We have previously shown that Bcl-3s activities can be modulated by the GSK-3 kinase. In addition to transcriptional regulation, this proteins activity can also be regulated by ubiquitination.